DNA-binding characteristics of the Escherichia coli CytR regulator: a relaxed spacing requirement between operator half-sites is provided by a flexible, unstructured interdomain linker.
The Escherichia coli CytR regulator belongs to the LacI family of sequence-specific DNA-binding proteins and prevents CRP-mediated transcription in the CytR regulon. Unlike the other members of this protein family, CytR binds with only modest affinity to its operators and transcription repression thus relies on the formation of nucleoprotein complexes with the cAMP-CRP complex. Moreover, CytR exhibits a rotational and translational flexibility in operator binding that is unprecedented in the LacI family. In this report we examined the effect of changing the spacing between CytR half-operators on CytR regulation in vivo and on CytR binding in vitro. Maximum repression was seen with the short spacing variants: repression peaks when the half-operators lie on the same face of the DNA helix. Repression was retained for most spacing variants with centre separations of half-operators < or = 3 helical turns. Our data confirm and extend the view that CytR is a highly flexible DNA binder that can adapt many different conformations for co-operative binding with CRP. Furthermore, limited proteolysis of radiolabelled CytR protein showed that the interdomain linker connecting the DNA binding domains and the core part of CytR does not become structured upon DNA binding. We conclude that CytR does not use hinge alpha-helices for minor groove recognition. Rather, CytR possesses a highly flexible interdomain linker that allows it to form complexes with CRP at promoters with quite different architecture.
Jørgensen
C I
CI
Department of Molecular Biology, Odense University, Odense M, Denmark.
Kallipolitis
B H
BH
Valentin-Hansen
P
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013463:Sulfuric Acid Esters / Q000737:chemistry; D001616:beta-Galactosidase / Q000032:analysis
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; C048425:CytR protein, E coli; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D013463:Sulfuric Acid Esters; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase; D010450:Endopeptidases; C007482:dimethyl sulfate